Abstract Recent phylogenetic analysis using mitochondrial cytochrome oxidase I (mtCOI) sequences of Bemisia tabaci worldwide indicates that the whitefly comprises at least 24 morphologically indistinguishable but genetically distinct cryptic species. While evidence of reproductive isolation has been reported for some of the putative species, more extensive crossing experiments are required to clarify the systematics of this species complex. In this study, we established laboratory cultures for six putative species of B. tabaci collected in China. We conducted 22 inter-species crosses among the six putative species. The data and those reported previously were collated, and the combined dataset covered all the 30 possible inter-species crosses among the six putative species. Intra-species controls always produced female and male progeny and the proportions of females in the first generation (F1) ranged from 56% to 70%. However, in inter-species crosses female progeny were rarely produced, and the few F1 females produced in four of the 30 inter-species crosses were either sterile or significantly weaker in viability. These results demonstrate a pattern of complete reproductive isolation among the six putative species and show that they are six cryptic species in the B. tabaci complex.